The FastLane Cell SYBR Green Kit accelerates and streamlines real-time RT-PCR and is ideal for experiments requiring rapid high-throughput gene expression analysis.The kit provides a set of buffers for preparing FastLane lysates from cultured cells. In addition to lysing cells, the buffers also stabilize cellular RNA and eliminate genomic DNA. FastLane lysates are used directly in SYBR Green-based real-time one-step RT-PCR. A variety of other detection formats are also available, including singleplex detection with sequence-specific probes or multiplex detection with sequence-specific probes (with or without ROX passive reference dye).

Using the FastLane Kit, A549 cells in a 384-well plate were analyzed for the expression of Eg5, GAPDH, HMBS, and Rab5. Data show the mean CT value with standard deviation from triplicate wells. (Data kindly provided by Anne Grabner, Cenix BioScience GmbH, Germany.)

Highly specific quantification.

Real-time one-step RT-PCR using the FastLane Kit and a QuantiTect Primer Assay for INPP5D (a phosphatase) enabled specific transcript quantification in HeLa, HepG2, and Cos cells, as shown by the single peaks in melting curve analysis.

Highly reproducible analysis of a 96-well plate.

Real-time one-step RT-PCR using the FastLane Kit and a QuantiTect Primer Assay for INPP5D (a phosphatase) gave reproducible CT values from a 96-well plate of HeLa ACC cells.

Specific transcript detection is guaranteed when the kit is used in combination with QuantiTect Primer Assays. These are predesigned, genomewide primer sets, available at GeneGlobe.

Procedure

With the FastLane Cell SYBR Green Kit, lysates are obtained in just 12 minutes in a 3-step procedure: cell wash; cell lysis with integrated RNA stabilization and genomic DNA elimination; and lysate pretreatment. After pretreatment, lysates are ready to use in real-time one-step RT-PCR.

The kit contains sufficient reagents for cell lysis in two 96-well cell culture plates and subsequent real-time RT-PCR (50 µl PCR volume). The kit can also be used to analyze a single 384-well cell culture plate if the PCR volume is reduced to 20 µl; if a 50 µl PCR volume is desired, an additional QuantiTect SYBR Green RT-PCR Kit (200) needs to be purchased.